In the presence of glucose, hemoglobin (Hb) is glycated by a nonenzymatic reaction stabilized by an Amadori rearrangement. Potential glycation sites are the N-terminal amino acids of the four polypeptide chains, and free .epsilon.-amino groups of lysine within the chains. The most reactive site, HbA1c, is the N-terminal valine of the .beta.chain, which accounts for about 60% of all bound glucose. Bound sugars can also appear at any of 44 glycation sites on the .epsilon.-amino groups within the chains (about 34% of clycations), and the N-terminus of the a-chains (about 6% of glycations). The rate of glycation at any one site is directly proportional to glycation at any other site. Therefore, there is a linear relationship between the total amount of glycated hemoglobin (GHb) and any particular form of glycated hemoglobin (e.g., HbA1c).
A determination of the level of GHb in an individual can serve as an index of the blood glucose levels in the blood over time. The measurement of GHb) in diabetics is a useful indicator of disease state. Because the average blood cell has a lifespan of 120 days, (GHb levels reflect the blood glucose history of an individual over several months (Singer, Ann. Intern. Med., 1989, 110:125-137), with most recent blood glucose levels being most strongly represented. It is recommended that diabetes be monitored by determining levels of GHb every 2-3 months to determine the progress of disease, the efficacy of control of the disease, or both.
Many methods for assessing GHb concentrations require separation of GHb from Hb. One type of separation method is based on the charge difference between GHb and Hb. This can be done, for example, by electrophoresis, high performance liquid chromatography (HPLC), or conventional ion exchange chromatography. Structural differences have also been exploited in assays of GHb, using methods such as affinity binding, chromatography, and immunoassay (Goldstein et al., Clin. Chem., 1996, 32/10B, B64-B70).
Antibodies that have limited specificity for native HbA1c have been obtained by using glycosylated synthetic peptide sequences from the N-terminal region of the Hb .beta.-chain (Knowles, U.S. Pat. Nos. 4,647,654 and 4,727,036; Mezei, U.S. Pat. No. 4,478,744). These antibodies have significantly less affinity for native HbA1c compared to the peptides against which they were raised. Because the antigenic site is difficult to access in native HbA1c, methods to denature HbA1c (Lewis, EP 401 860 A2; Knowles U.S. Pat. No. 4,727,036) have been used to develop immunoassays for HbA1c. Another approach is to degrade the Hb molecule with an enzyme, e.g., pepsin, to expose the N-terminal .beta.-chain site. Methods for immunoassay of GHb generally employ denaturation or degradation pretreatment steps for antibody and/or assay development.